For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecu
Trang 1on the outcome of intramolecular homologous
recombination
Paula Barros*, Francisco Boa´n*, Miguel G Blanco and Jaime Go´mez-Ma´rquez
Departamento de Bioquı´mica e Bioloxı´a Molecular, Facultade de Bioloxı´a-CIBUS, Universidade de Santiago de Compostela, Spain
The integrity of chromosomal material is dependent
upon the efficient repair of DNA double-strand breaks
(DSBs), which arise during DNA replication or are
caused by exogenous agents Without such systems,
unrepaired breaks can lead to chromosomal
transloca-tions, loss of transcriptional control, and promotion of
tumorigenesis [1] In human cells, the repair of DSBs
can take place through two independent systems:
homologous recombination (HR), and nonhomologous
end-joining HR is promoted by several enzymes of the
RAD52 epistasis group, which includes RAD51, the
human homolog of Escherichia coli RecA [2,3] This
protein promotes the key homologous pairing and strand-exchange reactions leading to the formation of interlinked recombination intermediates [4]
Changes in ionic strength alter the behavior of some enzymes involved in the HR process In this regard, previous work has shown that high salt concentrations provoke conformational changes in the RAD51 pro-tein [5] favoring the coaggregation of RAD51–ssDNA nucleoprotein filaments with duplex DNA, stimulating the recombination [6] More recently, a study defining the effect of salt on human RAD51 activities was reported [7] However, as far as we know, none of the
Keywords
G-quadruplex; minisatellite MsH43;
monovalent cations; recombination fidelity;
repetitive sequences
Correspondence
J Go´mez-Ma´rquez, Departamento de
Bioquı´mica e Bioloxı´a Molecular, Facultade
de Bioloxı´a-CIBUS, Universidade de
Santiago de Compostela, 15782 Santiago de
Compostela, Spain
Fax: +34 9815969054
Tel: +34 981563100 (ext 16937)
E-mail: jaime.gomez.marquez@usc.es
*These authors contributed equally to this
work
(Received 17 February 2009, revised 18
March 2009, accepted 20 March 2009)
doi:10.1111/j.1742-4658.2009.07013.x
Homologous recombination is a very important cellular process, as it pro-vides a major pathway for the repair of DNA double-strand breaks This complex process is affected by many factors within cells Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure
of DNA For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombina-tion, each with two copies of different alleles of the human minisatellite MsH43 We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+ We observed that the MsH43 recombi-nants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5¢-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences
Abbreviations
DSB, double-strand break; HR, homologous recombination.
Trang 2studies on the influence of salts on the recombination
process have analyzed how monovalent cations could
affect the fidelity of recombination, defined as the
per-centages of equal and unequal recombinant molecules,
and the frequency of recombination
In our laboratory, we have developed an in vitro
sys-tem with which to analyze HR and nonhomologous
end-joining [8–11] This system allows us to establish
in vitro the recombinogenic capacity of any DNA
sequence, as well as to determine the nature of the
recombinant molecules generated In the present study,
we employed this in vitro system to analyze the effect
of the monovalent cations K+, Na+ and NH4+ on
recombination For this purpose, we used the
minisat-ellite MsH43, a human DNA sequence composed of
pentamers and hexamers organized in a tandem array
[12,13] The organization in tandem of small repeat
units provides a good substrate with which to study
the frequency of equal and unequal crossovers, as it
facilitates perfect and nonperfect pairings We found
that the presence of monovalent cations led to a higher
proportion of equal recombinants, and hence an
increase in the fidelity of the recombination events in
the experimental system employed
On the other hand, it is well known that
guanine-rich nucleic acids (DNA and RNA) are capable of
forming four-stranded structures named
G-quadruplex-es (also known as G-tetrads, G4s, or G-quartets)
[14,15] These structures are further stabilized by the
presence of a monovalent cation (especially K+) in the
center of the tetrad [15,16] G-quadruplex-forming
sequences have been identified in eukaryotic telomers,
as well as in gene promoters, recombination sites, and
DNA tandem repeats [15] Whether or not genomic
G-rich structures can form quadruplex-based structures
in vivoremains to be fully demonstrated, although
sup-portive data are starting to emerge [14,15,17,18]
G-quadruplexes have long been hypothesized to play
roles in DNA recombination Thus, G-quadruplex
DNA might play a role in class switch recombination
in the immunoglobulin genes [19,20], and studies
in yeast suggest possible roles for G-quadruplex DNA
in homologous recombination during meiosis [21] In
relation to this, Hop1 not only binds to and catalyzes
the formation G-quadruplex DNA in vitro, but also
promotes the pairing of dsDNA molecules via
quadru-plex structures [22] However, is not yet clear how the
in vitroactivities of this and other proteins on
G-quad-ruplex DNA relate to their in vivo functions
In the present work, we also analyzed the effect of
the presence of sequences capable of forming
G-quad-ruplex structures on recombination frequency and the
generation of recombinant molecules, taking advantage
of the capacity of the minisatellite MsH43 to form this kind of structure in the presence of K+[12] We found that the presence of G-quadruplex did not alter the recombination frequency as compared with the allele control Moreover, the great majority of recombinants containing duplications generated in the presence of
K+ did not include the repeats located at the 5¢-side
of the G-quadruplex motif of MsH43, suggesting that this structure is involved in the recombination events leading to duplications A model to explain this find-ing, involving replication slippage, is also shown
Results and discussion
To study the effects of salts on the frequencies of equal and unequal recombinant products generated in the recombination experiments, we employed the mini-satellite MsH43, as it shows two useful features: (a) an organization in tandem, which allows the existence of different types of homologous pairings (in register or not in register), leading to the formation of equal and unequal recombinant molecules; and (b) the ability of allele 80.1 to form a G-quadruplex, as it contains the motif (TGGGGC)4, which is a G-quadruplex-forming structure, and the inability of allele 73.1 to form such
a structure, because it contains the motif TGGGGC repeated only three times instead of four [12]; this dif-ferential characteristic allows analysis of the effect of the presence of G-quadruplex structures on the genera-tion of recombinant molecules in the in vitro system employed in this work
To carry out the recombination analyses, we employed an in vitro system designed to detect intra-molecular homologous recombination events [8–11]
We constructed two pBR322-based plasmids, the recombinant substrates p73.1 and p80.1, bearing two copies cloned in the same orientation as the corre-sponding MsH43 allele, 73.1 or 80.1 The map of these recombinant substrates is shown in Fig 1A As the lacZ gene is situated between the two copies of the MsH43 inserts, the recombination that takes place after pairing of the MsH43 homologous sequences leads to the excision of the lacZ gene from the original substrate, generating two kind of recombinants, equal and unequal (Fig 1B) In the equal recombinants, the minisatellite remains unaltered, whereas in the unequal ones, the minisatellite displays size variations caused
by unequal pairings or alterations during the recombi-nation process
In the recombination experiments, each plasmid substrate was incubated with the nuclear extract under standard conditions [8] After incubation, DNA was extracted and used to transform bacteria The
Trang 3recombi-nant plasmids generated lacZ) bacteria (white
colo-nies), and the original plasmids generated lacZ+
bacte-ria (blue colonies) The recombinant plasmids were
first analyzed by restriction with EcoRI The digestion
of p73.1 recombinant products yielded two restriction
fragments, one with the minisatellite sequence, and the
digestion of the p73.1 original plasmid generated five
restriction fragments, two of them identical (those
con-taining the minisatellite) (Fig 2A) If the recombinant
was unequal, then the EcoRI fragment containing the
minisatellite showed size variations (asterisks in
Fig 2A) In the case of the p80.1, the digestion with
EcoRI of the original plasmid yielded four DNA
frag-ments, whereas the digestion of its recombinant
pro-ducts generated only one fragment (Fig 2B) To
facilitate the identification of p80.1 recombinants, the
recombinant plasmids were amplified by PCR with the
primers P02.1 and P02.2 [12] The analysis of
amplifi-cations products allowed differentiation of the majority
of the equal and unequal events (Fig 2C) However,
when the variation affected few repeats, it was difficult
to distinguish length variations of the minisatellite To
solve this problem, heteroduplex analyses [13] were
carried out by mixing the amplification products of
each recombinant with the PCR product obtained
from the MsH43 sequence present in the original
con-struct Figure 2D shows the result of a heteroduplex
assay employing the recombinants generated in the
experiments with p80.1 The generation of
hetero-duplex molecules denotes the presence of an unequal
recombinant, whereas the absence of heteroduplex molecules means that it is an equal recombinant The sequencing of 14 equal recombinants corroborated that the recombinant molecules classified as equal con-served the original minisatellite sequence (data not shown)
The results of the intramolecular homologous recombination experiments are summarized in Table 1 The data were collected from three indepen-dent experiments for each assay condition (standard
or supplemented with salts) The LacZ+ colonies were produced by the transformation with the origi-nal plasmids, whereas the LacZ) colonies were the result of transforming bacteria with the recombinant plasmids The lacZ) colonies due to mutations in the lacZ gene made up less than 1% of the total lacZ) colonies, and the frequency of the lacZ) colonies in transformations with the original substrate plasmids not exposed to the nuclear extract or with heat-inacti-vated extract (15 min, 100C) was about 2 · 10)5 Under standard conditions, both the recombination frequencies and the frequency of equal and unequal recombinants were very similar with plasmids p73.1 and p80.1 Noteworthy, with both plasmids, the recombination events that generated unequal recombi-nants were more abundant ( 20%) than those that maintained the original sequence of MsH43 To verify the effect of the presence of G-quadruplex-forming sequences in this in vitro system, we performed several assays in the presence of 20 mm K+, added to
stabi-Pairing of homologous sequences
X ori
MsH43
MsH43
LacZ
ori Amp
Crossover
ori
MsH43
Recombinant product
(LacZ –)
Equal recombinant
Unequal recombinant
p73.1 MsH43 73.1
MsH43 73.1
E E
E
E E
MsH43 73.1
E
MsH43 80.1
ori
p80.1 MsH43 80.1
MsH43 80.1 E
E
ori
Recombination substrates
Rec
substrate
A
B
Fig 1 Map of the recombination substrates
and representation of an intramolecular
homologous recombination event (A)
Plas-mids p73.1 and p80.1 (not drawn to scale)
were used as substrates in the
recombina-tion assays They contain a replicarecombina-tion origin
(ori), an ampicillin resistance gene (Amp),
the lacZ gene, and two identical copies of
the MsH43 sequence (wide black arrows),
cloned in the same orientation At the top is
shown a scheme of the MsH43 inserts,
minisatellite (open box), and flanking
sequences; thin arrows mark the position of
primers P02.1 and P02.2 E, EcoRI; Sc,
SacII; S, SalI (B) The intramolecular
homolo-gous recombination generates two kinds of
plasmid: equals, in which the MsH43
remains unaltered, and unequals, with
alter-ations in the minisatellite sequence.
Trang 4lize the G-quadruplex structure Once again, the
recombination frequencies were very similar with both
plasmids, indicating that the capacity of the MsH43
80.1 allele to form G-quadruplex does not influence the recombination frequency, at least in our in vitro system However, the presence of K+caused a strong
1 3 5 2 4 6 8 10 p80.1 12 13 14 7 9 11 15 16 21 17 18 19 20 22 23
1 3 5 2 4 6 8 7 M 9 10 11 12 13 14 15 16 17 18
* * *
*
* * *
*
*
A
B
C
D
Fig 2 Analysis of the recombinant prod-ucts generated in the in vitro recombination assays (A) Analysis in 1.5% agarose gels of the EcoRI digest of the recombinant plas-mids obtained from lacZ)colonies in experi-ments carried out with p73.1 The EcoRI restriction pattern of the original plasmid p73.1 (lane p73.1) and the recombinant products (lanes 1–15) are shown The arrow indicates the DNA fragment that contains the original MsH43 sequence, and asterisks mark the DNA fragment containing altera-tions in the size of MsH43 (unequal recomb-inants) (B) Analysis in 1.5% agarose gels of the EcoRI digest of the plasmids obtained from lacZ)colonies in recombination experi-ments carried out with p80.1 The EcoRI restriction patterns corresponding to the original plasmid p80.1 (lane p80.1) and the recombinant products (lanes 1–9) are shown (C) Analysis in 2% agarose gels of the amplification products of recombinant plasmids obtained in experiments carried out with p80.1 The amplification product of the original plasmid p80.1 (lane p80.1) and the recombinant products (lanes 1–23) are shown The arrow indicates the DNA frag-ment that contains the original MsH43 sequence, and asterisks mark unequal recombinants (D) Heteroduplex analysis in
a 5% polyacrylamide gel (the presence of heteroduplex molecules denotes the pres-ence of an unequal recombinant); lane p80.1
is shown as a control of no heteroduplex formation The arrow indicates the DNA fragment that contains the original MsH43 sequence M, 100 bp ladder (Promega).
Trang 5reduction (near 50%) in the recombination
frequen-cies, as well as an important decrease in the nuclease
activity of the nuclear extract (data not shown) As
the initiation of homologous recombination is
medi-ated by a nuclease activity that introduces DNA
DSBs [23], it is possible that the reduction in
recom-bination frequency was due to inhibition of the
nucle-ase activity by K+
Remarkably, in the presence of K+, the proportions
of equal and unequal recombinants were inverted with
respect to the results obtained under standard
condi-tions; that is, the equal recombinants were more
abun-dant ( 25%) than the unequal ones (Table 1) Was
this inversion produced specifically by K+? The
recom-bination assays carried out in the presence of Na+or
NH4+, maintaining the same ionic strength, showed a
marked reduction of the recombination frequency with
respect to the standard conditions, more pronounced
than with K+, and also a predominance of equal
rec-ombinants (Table 1) The finding that Na+and NH4+
caused a greater decrease in the nuclease activity of the
nuclear extract than K+ (data not shown) provides a
coherent explanation of the observation that the
recombination frequencies obtained with those cations were lower than with K+
Cations play essential roles in nucleic acid and pro-tein structure, stability, folding, and catalysis By means of their interactions with DNA and proteins, they could play an important role in recombination For instance, changes in K+concentration could alter chromatin structure by taking advantage of the unique sensitivity of quadruplex formation to K+ and other cations present in the cells [24,25] On the other hand,
in vitro studies with G-rich telomeric DNA sequences and the minisatellite MsH43 have shown that they can form quadruplex structures whose stability is sensitive
to changes in the concentrations of important physio-logical cations such K+ [12,16] As mentioned earlier, RAD51, a key enzyme in HR, is affected by salts Our results provide strong evidence that the presence of monovalent cations causes a strong decrease in recom-bination frequency, probably due to inhibition of the nuclease activity that produces DSBs on the plasmid substrates, and leads to enhancement of the fidelity of recombination, as the proportion of equal recombi-nants was higher The presence of monovalent cations
Table 1 Quantitative analysis of recombination experiments The recombinant frequencies are given as the ratio between the number of LacZ)colonies and the total number of colonies obtained in each assay The equal and unequal recombinant frequencies are given as the ratio between the total number of each type of recombinant and the total number of recombinants For columns 4, 6 and 8, mean ± stan-dard deviation of the data is provided.
Recombination
substrate
Assay conditions
Colonies (LacZ + ⁄ LacZ))
Recombination frequency (%)
Equal recombinants Unequal recombinants
No Frequency (%) No Frequency (%)
Trang 6in the nucleus of the cells is an important physiological
requirement, and our results suggest that monovalent
cations also influence genomic stability through their
participation in the recombination process This
increase in fidelity could be related to alterations in the
structure of the minisatellite and to conformational
changes in proteins involved in recombination In this
regard, it has been reported that high salt induces
con-formational changes in RAD51, leading to the
forma-tion of interlinked recombinaforma-tion intermediates [2] that
are essential for the correct progression of the
recom-bination process It is worth noting that, with the
in vitrosystem developed in our laboratory, we did not
observe an influence of the capacity to generate
G-quadruplex DNA on the recombinogenic frequency
of the minisatellite MsH43
The existence of unequal recombinants allowed a search for the sites where the rearrangement in the MsH43 occurred The analysis of 163 recombinant sequences (Figs 3 and 4) revealed that duplications occurred less frequently (30%) than deletions, similar
to what was reported for the minisatellite CEB1 [26,27], suggesting that this type of repetitive sequence
is prone to undergoing deletions in the recombination process Most of the unequal recombinants involve simple deletions (Fig 3), and only four recombinants derived from p80.1 (S5, S11, K10 and K13 in Fig 3A) showed double deletions, suggesting that they did not arise by a simple recombination event With regard to the MsH43 expansions, they seem to be the conse-quence of simple direct duplications (Fig 4), except in one case derived from p73.1, where one repeat was
Fig 3 Organization of MsH43 recombinants containing deletions (A) Sequence array of the deleted molecules obtained in experiments with p80.1: standard conditions (S1–S18), with K + (K1–K21), with Na + (Na1–Na9), and with NH4+ (N1–N4); asterisks indicate recombinants with double deletions The sequence of the MsH43 80.1 allele is shown (B) Sequence array of the deleted molecules obtained in experiments with p73.1: standard conditions (S1–S17) and with K + (K1–K19) The sequence of the MsH43 73.1 allele is shown The discontinuity in the sequence indicates the deleted fragment Minisatellite repeats are depicted by a color code shown at the bottom.
Trang 7intercalated between the duplicated fragments (S5 in
Fig 4B) None of the recombinants analyzed showed
truncated repeats This feature was also observed in
the recombinants generated by the human minisatellite
MsH42 [8,9] and by the human minisatellite CEB1
inserted in yeast [26,27], indicating that the
reorganiza-tions produced in the minisatellite MsH43 arose by a
homology-guided mechanism Interestingly, in the
presence of K+, the p80.1 recombinants displaying
duplications did not include the repeats located at the
5¢-side of the G-quadruplex motif (K1–K14 in
Fig 4A) In contrast, this limitation was not found in
the assays carried out either under standard conditions
or in the presence of Na+ or NH4+, or with p73.1
(Fig 4) It is tempting to speculate that the
G-quadru-plex motif (TGGGGC)4 influences the resolution of
the recombination events, leading to duplications in the minisatellite sequence
There is an emerging consensus that secondary structures of DNA have the potential to induce geno-mic instability The role of nonlinear DNA in replica-tion, recombination and transcription has become evident in recent years Several studies have predicted and characterized regulatory elements at the sequence level However, little is known about the role of DNA structures as regulatory motifs Cells use these struc-tural motifs as signals for processes such as gene regu-lation or recombination in both prokaryotes and eukaryotes [28,29] The coincidence of breakpoints of gross deletions with non-B DNA conformations has led to the conclusion that these structures can trigger genomic rearrangements through recombination⁄ repair
S1
S3
S2
S4
S6
S5
S8
S9
S7
S10
S12
S11
S13
K1
K3
K2
K4
K6
K5
K8
K9
K7
K10
K12
K11
K13
K14
Na1
Na3
Na2
Na4
Na6
Na5
Na7
N1
N3
N2
N4
MsH43 80.1
allele
N5
N6
N7
MsH43 73.1 allele S1 S3 S2 S4 S6 S5
S8 S9 S7
S10 S12 S11 S13 S15 S14
K1 K3 K2 K4 K6 K5
K8 K9 K7
K10 K12 K11 K13 K15 K14 K16 K18 K17 K19
Fig 4 Organization of MsH43 recombinants containing duplications (A) Sequence array of recombinants with duplications obtained in experiments with p80.1: standard conditions (S1–S13), K + (K1–K14), Na + (Na1–Na7), and NH4+ (N1–N7) The sequence of the MsH43 80.1 allele is shown (B) Sequence array of recombinants presenting duplications obtained in experiments with p73.1 under standard conditions (S1–S15) and with K + (K1–K19) The sequence of the MsH43 73.1 allele is shown The marks in the recombinant S1 denote the mutations with respect to the original MsH43 allele The arrowhead indicates the recombinant that has a repeat intercalated between the duplicated arrays Arrows indicate the duplicated fragment.
Trang 8activities [30] Furthermore, G-quadruplex secondary
structures can induce genetic rearrangements and
pro-mote RecA-independent homologous recombination
[31] Genome-wide predictions have shown an
abun-dance of G-quadruplex DNA motifs in the genomes of
Homo sapiens[32] and E coli [33] In both species, the
distribution of G-quadruplex structures seems to be
nonrandom and linked to regulatory regions of the
genome The important finding is that this kind of
structure may play a role in genome dynamics at three
levels: regulation of transcription, recombination and
mutation hotspots in vivo, and blocking the
progres-sion of DNA polymerases [34,35]
Our results demonstrate that a minisatellite sequence,
which is not included inside a gene [12], can form
G-quadruplex structures that interfere with DNA synthesis
and influence the resolution of recombination It is
tempting to speculate that this type of repetitive
sequence could be involved in processes related to
gen-ome stability In this regard, although the mechanisms
involved in minisatellite instability are poorly
under-stood, some relevant factors have already been found,
such as the requirement for DSBs [23] and length and
sequence heterozygosity [36] Furthermore, size
altera-tions of G-rich minisatellites can be caused by the ability
of these sequences to adopt G-quadruplex structures
[37] and by their capacity to undergo slippage during
replication or unequal crossovers [8,9,38] In the case of
MsH43, we observed that the recombinants containing
duplications, generated in the presence of K+, did not
include the repeats located at the 5¢-side of the
G-quad-ruplex motif of MsH43, suggesting that this structure is
involved in the recombination events leading to
duplica-tions In Fig 5, we show a hypothetical model to
explain the mechanism involved in the generation of
duplications in the presence of a G-quadruplex
Accord-ing to this, the generation of duplications is explained
by replication slippage on the strand of new synthesis
The presence of a G-quadruplex structure stabilized by
K+in the slippage loop would interfere with the
replica-tion at the 5¢-end of the G-quadruplex motif and
conse-quently with the generation of duplications containing
this 5¢-region of MsH43 This effect is not observed if
the slippage occurs either on the 3¢-side of the
G-quad-ruplex DNA structure or in the template strand; in the
latter case, the slippage would produce the observed
deletions This model explains the generation of all
duplications derived from the experiments with p80.1 in
the presence of K+, with the exception of K1, which
contains the G-quadruplex motif in the duplicated
sequence (Fig 4A) Possible explanations for
the generation of the recombinant K1 could be that not
all p80.1 plasmid molecules present in the
recombina-tion assay formed G-quadruplex, or that this was unstable In both cases, the slippage process would not be interfered with by the G-quadruplex structure, making possible the inclusion of the sequence (TGGGGC)4in some recombinant molecules Support-ing this reasonSupport-ing are the results found in dimethyl sulfate methylation protection assays carried out with oligonucleotides designed from the sequence of several MsH43 alleles [12] In these experiments, even at con-centrations of 100 mm K+, there is a residual amount of oligonucleotides that do not form G-quadruplex The present work shows that the presence of mono-valent cations increases the fidelity of recombination, and that this effect is independent of the presence of G-quadruplex structures in the minisatellite MsH43 However, the G-quadruplex structure seems to be a barrier to the events leading to duplications, perhaps leading to blockage of polymerases at that point Therefore, the G-quadruplex would not be a stimulus for recombination but a source of genomic instability
5´
5´
5´
(TGGGGC)4
5´
5´
5´
3´
DSB
A
B C
G4 structure
Fig 5 Recombination model involving G-quadruplex (G4) structure and slippage processes for the generation of recombinant mole-cules containing duplications and deletions (A) Generation of a DSB by the nuclease(s) of the nuclear extract initiates the recombi-nation process; the sequence (TGGGGC) 4 represents the G-quadru-plex motif in the MsH43 80.1 allele (B) After the break, there is a strand invasion of the homologous sequence in the second copy of the minisatellite included in the recombinant plasmid employed in the assay (C) Replication slippage may occur in the strand of new synthesis (left side) If the slippage loop comprises the region con-taining the G-quadruplex motif, a G-quadruplex structure could be generated that would be stabilized by the presence of K+; this sec-ondary structure would interfere with DNA synthesis, avoiding the formation of duplications involving the 5¢-side of the G-quadruplex motif When the slippage loop is located at the 3¢-side of the G-quadruplex DNA motif [right side of (C)], duplications can be gen-erated without the interference of G-quadruplex structures Accord-ing to this model, it is worth notAccord-ing that the slippage in the template strand, leading to deletions in the MsH43 sequence, would not be affected by the generation of G-quartets, as it does not have the G-quadruplex DNA motif.
Trang 9It should be noted that the results obtained with
MsH43 cannot be applied to any DNA sequence, as
the repetitive nature of MsH43 favors the existence of
unequal crossovers as well as slippage processes
Perhaps one of the functions of repetitive DNA in the
genomes is to serve as instability spots that are
neces-sary for genome evolution
Finally, the results presented here show that the
in vitro system used in this study may be useful for
investigation of the mechanisms involved in
recombi-nation and DNA instability, as well for the analysis of
how monovalent cations affect the proteins implicated
in this fundamental biological process
Experimental procedures
Recombination substrates
The alleles of MsH43 used in this study, 73.1 and 80.1,
were obtained by amplification of human genomic DNA,
with the primers P02.1 and P02.2 [10] The PCR products
were cloned in the pGEM-T Easy vector (Promega,
Madi-son, WI, USA) The plasmid containing the 73.1 allele was
digested with SacII–SalI, generating a 569 bp fragment,
and the plasmid containing the 80.1 allele was digested with
EcoRI, producing a 586 bp fragment To generate the
recombination substrates, plasmids p73.1 and p80.1, two
identical copies of each fragment were cloned in pBR322,
in the same orientation, flanking the lacZ gene (Fig 1A)
In vitro recombination assays
We have previously shown that the recombination
prod-ucts are generated by the nuclear extract and not by the
repair machinery of bacteria [7] The standard
recombina-tion reacrecombina-tions were performed in a final volume of 100 lL
containing 20 mm Tris⁄ HCl (pH 7.5), 10 mm MgSO4,
1 mm ATP, 0.1 mm each dNTP, 1 lg of plasmid (p73.1 or
p80.1), and 10 lg of rat testis nuclear extract [6] In the
experiments containing KCl, NaCl, or NH4Cl, the salts
were added to a final concentration of 20 mm Several
concentrations of salts were assayed (5, 10, 15, 20, and
25 mm), and a concentration of 20 mm was chosen for the
recombination experiments, because it produces a good
number of white colonies, allowing the screening of many
recombinants per assay (data not shown) Concentrations
higher than 20 mm produced few white colonies, probably
because of the decrease caused in the recombination
fre-quency, which could be due to inhibition of nuclease
activity The inhibition of nuclease activity was observed
by the analysis of plasmid integrity, after incubation with
the nuclear extract in the presence of the different
mono-valent cation concentrations, by electrophoresis on agarose
gels After incubation of recombination substrates with the
nuclear extracts for 30 min at 37C, DNA was phenol-extracted, ethanol-precipitated, and used to transform
E coli DH5a cells Bacteria were plated onto LB agar plates containing Blue-O-Gal (BRL, Gaithersburg, MD, USA) at 0.3 gÆL)1 as lacZ gene indicator We observed that lacZ) colonies due to mutations in the lacZ gene made up less than 1% of the total lacZ)colonies The fre-quency of the lacZ) colonies in transformations with the original substrate plasmids not exposed to the nuclear extract or with heat-inactivated extract (15 min, 100C) was about 2· 10)5 The white colonies (recombinant products) were used for minipreparation of plasmid DNA and aliquots of 300 ng were digested with EcoRI, and analyzed in agarose gels
PCR, DNA sequencing, and heteroduplex analysis
The PCR reactions were performed in 25 lL containing PCR buffer [67 mm Tris⁄ HCl, pH 8.8, 16 mm (NH4)2SO4, 0.01% Tween-20], 0.1 ng of plasmid, 0.3 lm each primer (P02.1 and P02.2), 0.2 mm dNTPs, 1.5 mm MgCl2, and 0.5 U of Taq polymerase Cycling conditions were 29 cycles
of 95C for 1 min, 56 C for 30 s, and 72 C for 40 s, and
a final cycle with an extension of 5 min When the PCR products were used for direct cycle sequencing employing the dGTP BigDye Terminator v3.0 Sequencing kit (Applied Biosystems, Foster City, CA, USA), they were treated with exonuclease I and alkaline phosphatase (Exo⁄ Sap-It) (USB, Cleveland, OH, USA) After this treatment, the PCR prod-ucts were cycle sequenced by 25 cycles of 96C for 10 s and 68C for 2 min in a PTC-200 thermocycler (MJ Research, Ramsey, MN, USA), and purified by ethanol precipitation The sequencing products were analyzed using the 377 DNA Automated Sequencer (Applied Biosystems) For the heteroduplex analysis, aliquots of 5 lL of the PCR products obtained from the recombinants and from the ori-ginal recombination substrates were mixed at 95C for
3 min, and slowly cooled to room temperature The hetero-duplex molecules were detected by electrophoresis in 5% polyacrylamide gels (29 : 1) at a constant voltage of 140 V for 6 h using 1· TBE buffer (0.09 m Tris ⁄ borate, 0.002 m EDTA) and visualized by ethidium bromide staining
Acknowledgements
This work was supported by the Spanish Ministerio de Educacio´n y Ciencia (BFU2006-06708) and by the Xunta de Galicia (PGIDT07PX12001099R)
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